Substrate selectivity of human aldehyde oxidase 1 in reduction of nitroaromatic drugs

Article abstract of DOI:10.1016/j.abb.2018.10.017

Human aldehyde oxidase 1 (AOX1) catalyzes the oxidation of various drugs and endogenous compounds. Recently, we found that AOX1 catalyzed the reduction of drugs such as nitrazepam and dantrolene. In this study, we aimed to clarify the substrate selectivit

Full text of DOI:10.1016/j.abb.2018.10.017

Accepted Manuscript
Substrate selectivity of human aldehyde oxidase 1 in reduction of nitroaromatic drugs
Takuo Ogiso, Tatsuki Fukami, Kenji Mishiro, Keigo Konishi, Jeffrey P. Jones, Miki
Nakajima
PII:
S0003-9861(18)30570-8
https://doi.org/10.1016/j.abb.2018.10.017
YABBI 7844
DOI:
Reference:
To appear in: Archives of Biochemistry and Biophysics
Received Date: 21 July 2018
Revised Date: 27 September 2018
Accepted Date: 23 October 2018
Please cite this article as: T. Ogiso, T. Fukami, K. Mishiro, K. Konishi, J.P. Jones, M. Nakajima,
Substrate selectivity of human aldehyde oxidase 1 in reduction of nitroaromatic drugs, Archives of
Biochemistry and Biophysics (2018), doi: https://doi.org/10.1016/j.abb.2018.10.017.
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ACCEPTED MANUSCRIPT
Substrate Selectivity of Human Aldehyde Oxidase 1 in Reduction of Nitroaromatic
Drugs
Takuo Ogisoa, Tatsuki Fukamia,b, Kenji Mishiroc, Keigo Konishia, Jeffrey P. Jonesd, and Miki
Nakajimaa, b
a Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa
University, Kanazawa, Japan
b WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Japan
c Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, Japan
d Department of Chemistry, Washington State University, Pullman, Washington
To whom all correspondence should be sent:
Tatsuki Fukami
Drug Metabolism and Toxicology
Faculty of Pharmaceutical Sciences
Kanazawa University
Kakuma-machi, Kanazawa 920-1192, Japan
Tel: +81-76-234-4438 / Fax: +81-76-264-6282
E-mail: tatsuki@p.kanazawa-u.ac.jp
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Abstract
Human aldehyde oxidase 1 (AOX1) catalyzes the oxidation of various drugs and
endogenous compounds. Recently, we found that AOX1 catalyzed the reduction of drugs such
as nitrazepam and dantrolene. In this study, we aimed to clarify the substrate selectivity of
human AOX1 for the reduction of nitroaromatic drugs to obtain helpful information for drug
development. We investigated whether 11 nitroaromatic drugs were reduced by AOX1 using
recombinant AOX1 and human liver cytosol (HLC) in the presence of N1-methylnicotinamide,
an electron donor to AOX1. We found that clonazepam, flunitrazepam, flutamide, nilutamide,
nimesulide, and nimetazepam were substantially reduced by recombinant AOX1 and HLC,
whereas azelnidipine, nifedipine, and nimodipine were slightly reduced and metronidazole
and tolcapone were not reduced. Via structural analysis, we observed that nitroaromatic drugs
reduced by AOX1 possessed a relatively electron-deficient nitro group. Since the addition of
NADPH to human liver microsomes (HLM) did not increase the reductase activities of the
drugs that were reduced by recombinant AOX1, it was determined that NADPH-dependent
enzymes in microsomes, such as cytochrome P450, were not involved in this process.
Inhibition studies using known AOX1 inhibitors supported the role of AOX1 in the reduction
of drugs in HLC. In conclusion, this provides new information related to the substrate
selectivity of human AOX1 for the reduction of nitroaromatic drugs.
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1. Introduction
Drug-metabolizing enzymes metabolize over 70% of the drugs in our body to increase
their hydrophilicity and promote their excretion. Drug metabolism reactions are classified into
phase I and phase II reactions, and phase I reactions include oxidation, reduction, and
hydrolysis. The aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR)
families are well-studied enzyme families that cause drug reduction. For example, AKR1C3
and carbonyl reductase 1 (CBR1) catalyze the reduction of doxorubicin [1,2] to detoxify it,
and CBR1 reduces loxoprofen to form a pharmacologically-active metabolite [3].
Aldehyde oxidase 1 (AOX1) is known to catalyze the oxidation of drugs such as
allopurinol and zaleplon in humans [4–6]. In recent years, AOX1 has received attention for its
role in drug metabolism [7,8]. We recently found that AOX1 catalyzes the reduction of the
aromatic nitro groups of nitrazepam and dantrolene to form arylamine metabolites [9,10]. In
this reduction process, hydroxylamines, which are known to cause hepatotoxicity via binding
to DNA or proteins [11] or reactive oxygen species [12], are likely formed as intermediates
[9,10] (Fig. 1). Additionally, hydroxylamines are produced via oxidation of arylamines by
enzymes, including cytochrome P450s [13] (Fig. 1). Therefore, the reduction of nitroaromatic
drugs can increase hepatotoxicity, and some nitro group-containing drugs have been known to
cause liver injury as a side effect [11].
Many nitroaromatic drugs are available on the market. Some of these drugs appear to be
reduced at their nitro group in the human body [14,15], prompting us to speculate that
nitroaromatic drugs other than nitrazepam and dantrolene can also be reduced by AOX1. In
this study, we investigate the potential of human AOX1 to reduce nitroaromatic drugs and
discuss the chemical structures of drugs reduced by AOX1.
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2. Materials and methods
2.1. Materials
Human liver cytosol (HLC) (pooled, n = 150) and human liver microsomes (HLM)
(pooled, n = 50) were obtained from Corning (Corning, NY). Azelnidipine, clonazepam,
dantrolene sodium salt, flunitrazepam, flutamide, nilutamide, nimetazepam, nitrazepam, and
nifedipine were purchased from Wako Pure Chemical Industries (Osaka, Japan).
Aminoflunitrazepam, aminonitrazepam, nimesulide, and nimodipine were purchased from
Sigma-Aldrich (St. Louis, MO). Tolcapone and metronidazole were purchased from Toronto
Research Chemicals (Toronto, Canada) and LKT Laboratories (St. Paul, MN), respectively.
N1-Methylnicotinamide (MNA) was purchased from Cosmo Bio (Tokyo, Japan).
Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and β-nicotinamide adenine
dinucleotide phosphate (NADP+) were purchased from Oriental Yeast (Tokyo, Japan).
Aminodantrolene, a reduced metabolite of dantrolene, was synthesized previously [10].
Recombinant AOX1 expressed in Escherichia coli was prepared previously [16]. All other
chemicals and solvents were of the highest grade commercially available.
2.2. Synthesis of reduced metabolites of nitroaromatic drugs
2.2.1. Aminoclonazepam, aminoflutamide, aminonilutamide and aminonimetazepam
Clonazepam (10.8 mg, 34.2 µmol), flutamide (20.0 mg, 72.9 µmol), nilutamide (30.2 mg,
95.2 µmol), or nimetazepam (4.6 mg, 15.6 µmol) dissolved in ethanol (5 mL) was incubated
for 12-87 hr with SnCl2 (29.5-200.4 mg), a reducing agent. After we confirmed that these
compounds were completely reduced via thin-layer chromatography (TLC), the reaction was
quenched with saturated NaHCO3 (aq., 3 mL). The formed amine was then extracted with
ethyl acetate. The organic phase was dried at 37˚C under a nitrogen gas stream and evaporated
with an oil pump. 1H NMR analysis was performed in CDCl3 with a JEOL JNM ECA400
(400 MHz) spectrometer to determine the structure of the obtained amine. The chemical shifts
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were shown in supplemental Table S1.
2.2.2. Aminonimesulide and aminotolcapone
Nimesulide (26.0 mg, 84.3 µmol) or tolcapone (10.7 mg, 39.2 µmol) were dissolved in
ethyl acetate (5 mL), which was mixed with 10% activated Pd/C (2.6 mg or 2.0 mg) and
stirred for 1 hr or 5 min in a hydrogen atmosphere. After we confirmed that these compounds
were completely reduced via TLC, the mixture was filtered through a pad of Celite to remove
the catalyst and dried at 37˚C under a nitrogen gas stream. The residue was dissolved in ethyl
acetate, and hexane was added to the ethyl acetate solution to form a precipitate. The
precipitate was collected, washed with diethyl ether, and dried under reduced pressure. 1H
NMR analysis was performed in CDCl3 with a JEOL JNM ECA400 (400 MHz) spectrometer
to determine the structure of the obtained amine.
2.2.3. Aminoazelnidipine, aminonifedipine, and aminonimodipine
Azelnidipine (5.9 mg, 10.1 µmol) or nimodipine (8.9 mg, 21.3 µmol) were dissolved in
ethyl acetate (5 mL), and nifedipine (14.2 mg, 41.0 µmol) was dissolved in methanol (5 mL);
these compounds were mixed with 10% activated Pd/C (2.0 mg) and stirred for 5 min-31 hr in
a hydrogen atmosphere. After we confirmed that these compounds were completely reduced
via TLC, the mixture was filtered through a pad of Celite to remove the catalyst and dried at
37˚C under a nitrogen gas stream. The formed amine was dried under reduced pressure. 1H
NMR analysis was performed in CDCl3 with a JEOL JNM ECA400 (400 MHz) spectrometer
to determine the structure of the obtained amine.
2.2.4. Aminometronidazole
Fifty microliters of 4 M HCl in ethyl acetate was added to metronidazole (25.0 mg)
dissolved in 1,4-dioxane (5 mL). The mixture was stirred for 10 min and concentrated in
vacuo. The residue was dissolved in methanol (1 mL) and stirred with 10% activated Pd/C
(25.0 mg) in a hydrogen atmosphere. After we confirmed that these compounds were
completely reduced via TLC, the mixture was filtered through a pad of Celite to remove the
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catalyst and mixture was dried at 37˚C under a nitrogen gas stream. The formed amine was
dissolved in methanol, and the precipitate that was formed by adding ethyl acetate was dried
under reduced pressure. 1H NMR analysis was performed in D2O with a JEOL JNM ECA400
(400 MHz) spectrometer to determine the structure of the obtained amine.
2.3. Reductase activities of nitroaromatic drugs by recombinant human AOX1, HLM, and
HLC
To measure the reductase activities exhibited toward the 11 nitroaromatic drugs
(azelnidipine, clonazepam, flunitrazepam, flutamide, metronidazole, nifedipine, nilutamide,
nimesulide, nimetazepam, nimodipine, and tolcapone), a typical incubation mixture (final
volume of 0.2 mL) containing 25 mM or 100 mM potassium phosphate buffer (pH 7.4), 67.5
nM recombinant human AOX1 or 0.5 mg/mL HLM or HLC, and in some cases, an
NADPH-generating system (0.5 mM glucose-6-phosphate, 5 mM MgCl2, and 1 U/mL
glucose-6-phosphate dehydrogenase) or 2 mM MNA was prepared. After preincubation at
37˚C for 2 min, reactions were initiated by addition of a substrate dissolved in DMSO at a
final concentration of 100 µM. The final concentration of DMSO was 1%. After a 3 hr
incubation at 37˚C, the reaction was terminated by the addition of 200 µL of ice-cold
acetonitrile. The mixture was centrifuged at 20,380 g for 5 min, and 5 µL of the supernatant
was subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis
using an LCMS-8040 (Shimadzu, Kyoto, Japan) equipped with an LC-20AD HPLC system.
The column used was a Develosil ODS-UG-3 (2.0 × 150 mm, 3 µm; Nomura chemical, Seto,
Japan). The flow rate was 0.2 mL/min, and the column temperature was 40˚C. The mobile
phase was (A) 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. Nitrogen
was used as the nebulizer gas and drying gas. The operating parameters were optimized as
follows: nebulizer gas flow, 3 L/min; drying gas flow, 15 L/min; desolvation line temperature,
300 ˚C; and heat block temperature, 400 ˚C. The LC-MS/MS was operated in the positive
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electrospray mode. Each arylamine, a reductive metabolite of nitroaromatic drug, was
monitored in multiple reaction monitoring (MRM) mode. The conditions of the mobile phase
and m/z values for the detection and collision energies (CE) are summarized in Table 1.
Analytical data were processed using LabSolutions (version 5.82.1; Shimadzu). Reductase
activities toward dantrolene and nitrazepam, which have been demonstrated to be substrates
of AOX1 in our previous studies [9,10], were also determined.
2.4. Kinetic analyses
Kinetic analyses were performed with the conditions summarized in supplemental Table
S2. The assay conditions were determined to maintain linearity with respect to protein
concentration and incubation time in advance. The kinetic parameters were estimated from a
curve fitted using a computer program designed for nonlinear regression analysis
(KaleidaGraph; Synergy Software, Reading, PA). The following equations were used:
Michaelis-Menten equation: V = Vmax * [S] / (Km + [S])
CLint = Vmax / Km
where V is the velocity of the reaction, S is the substrate concentration, Km is the
Michaelis-Menten constant, and Vmax is the maximum velocity.
2.5. Inhibition studies
The reductase activities of nitroaromatic drugs in HLC were measured in the presence of
50 µM estradiol, estrone, and raloxifene (potent inhibitors of AOX1, 93 – 100% inhibition or
IC50 values 0.0057 – 0.43 µM against phthalazine oxidation in HLC), ketoconazole,
promazine, and simvastatin (moderate inhibitors of AOX1, 79 – 87% inhibition), diclofenac,
metronidazole, and naloxone (non-inhibitors of AOX1, 0 – 9% inhibition) [17,18], or
allopurinol [xanthine oxidoreductase (XOR) inhibitor]. The final concentration of
nitroaromatic drugs were set at approximately the Km values obtained using HLC as follows;
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clonazepam, nitrazepam: 25 µM, flunitrazepam, dantrolene: 60 µM, flutamide: 50 µM,
nilutamide: 200 µM, nimesulide: 250 µM, nimetazepam: 40 µM. The final concentration of
DMSO in the incubation mixture was 2%. The other conditions were same as described
above.
2.6. Statistical analysis
Statistical analyses were performed using one-way analysis of variance (ANOVA) and
Tukey's test. A value of P < 0.05 was considered statistically significant.
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3.
Result and Discussion
3.1. Reduction of nitroaromatic drugs by recombinant human AOX1
We investigated whether nitroaromatic drugs other than nitrazepam and dantrolene could
be reduced by AOX1. The nitroaromatic drugs azelnidipine, clonazepam, flunitrazepam,
flutamide, metronidazole, nifedipine, nilutamide, nimesulide, nimetazepam, nimodipine, and
tolcapone were used. We chemically synthesized the corresponding reduced amino forms of
these drugs as standards. These 11 nitroaromatic drugs were incubated with recombinant
human AOX1 in the presence of MNA, which is a substrate of AOX1 to be oxidized and
transfers electrons to AOX1 by its oxidation to enhance reduction of nitroaromatic
compounds (Fig. 2). We found that 6 drugs (clonazepam, flunitrazepam, flutamide, nilutamide,
nimesulide, and nimetazepam) were reduced by AOX1, whereas 3 drugs (azelnidipine,
nifedipine, and nimodipine) were slightly reduced and metronidazole and tolcapone were not
reduced. We confirmed that dantrolene and nitrazepam were also reduced by AOX1 as
positive controls [9,10]. Thus, it was demonstrated that not all nitroaromatic drugs were
reduced by AOX1.
3.2. Reduction of nitroaromatic drugs in HLM or HLC
We next investigated whether the 11 nitroaromatic drugs were reduced in HLC, where
AOX1 is localized, or in HLM, which has other reductases present (Fig. 3). The reductase
activities were detected in HLC for the 6 drugs that were reduced by recombinant human
AOX1, and their reduction was significantly increased by the addition of MNA (Figs. 3A - F).
Similar results were observed for the reduction of dantrolene and nitrazepam (Figs. 3G and H).
These results suggest that the reductase activities in HLC are largely due to AOX1. Reductase
activities were detected in HLC even in the absence of MNA, which may be due to the
presence of a contaminant MNA or other endogenous electron donors for AOX1 in HLC.
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The addition of NADPH in HLC did not increase the reductase activities toward
clonazepam, flutamide, and nilutamide (Figs. 3A, C, and D), whereas it did increase the
reductase activities toward flunitrazepam, nimesulide, and nimetazepam (Figs. 3B, E, and F).
The increase in the activities of the latter group may imply the involvement of
NADPH-dependent reductases, such as AKR enzymes, SDR enzymes, or NADPH-quinone
oxidoreductase 1 (NQO1), although it remains unclear whether these enzymes can catalyze
the reduction of nitro groups. The reductase activities related to clonazepam, flunitrazepam,
flutamide, nilutamide, nimesulide, and nimetazepam in HLM were exceptionally lower than
those observed in HLC and were not increased by NADPH (Figs. 3A - F), indicating that the
contribution of cytochrome P450, a major drug-metabolizing enzyme that requires NADPH,
is negligible.
Azelnidipine, metronidazole, nifedipine, nimodipine, and tolcapone, which were slightly
reduced or not reduced by recombinant AOX1, were barely reduced in either HLC or HLM
(Figs. 3I - M). Thus, it was demonstrated that for the nitroaromatic drugs analyzed in this
study, their reduction in the human liver could be largely attributed to AOX1.
3.3. Kinetic analyses of the reductase activities in HLC
Kinetic analyses of the reduction of clonazepam, flunitrazepam, flutamide, nilutamide,
nimesulide, and nimetazepam in HLC were performed in the presence or absence of MNA
(Figs. 4A - F and Table 2). The kinetics of all of the examined drugs were fitted to a
Michaelis-Menten kinetics curve. The Vmax values of all of the drugs were significantly
increased by MNA, likely due to an enhancement of the AOX1 activity. A similar increase
was observed in the reduction of dantrolene and nitrazepam (Figs. 4G and H, Table 2). The
Km values of the flunitrazepam, flutamide, nimesulide, and nimetazepam reductase activities
in the presence of MNA were similar to those in the absence of MNA, whereas the Km values
of clonazepam and nilutamide were increased by MNA. The increase in the Km values may
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be due to structural changes in the substrate binding site of AOX1 induced by MNA via an
allosteric effect because the substrate binding site of AOX1 for reduction appears to be
different from that observed for oxidation [19]. Collectively, the significant increase in the
catalytic efficiency in the presence MNA supported the ability of AOX1 to reduce
nitroaromatic drugs in the human liver.
3.4. Inhibitory effects of AOX1 inhibitors on the reductase activity of 6 nitroaromatic drugs
in HLC
To investigate whether the reduction of 6 nitroaromatic drugs in the human liver was
catalyzed by AOX1, not other enzymes that require MNA, an inhibition study was performed
at Km values of nitroaromatic drugs in HLC. The used inhibitors (potent inhibitors, moderate
inhibitors, and non-inhibitors of AOX1) were from papers by Obach (2004) [18] and Obach et
al. (2004) [17]. If the inhibitory potency was observed as reported, the responsibility of
AOX1 for the reduction of 6 nitroaromatic drugs in HLC could be explained. As a result, the
reductase activities of all of the tested drugs were potently inhibited by estradiol, estrone, and
raloxifene and were moderately inhibited by ketoconazole, promazine, and simvastatin,
whereas the reductase activities were barely inhibited by diclofenac, metronidazole, and
naloxone (Figs. 5A - F). Similar inhibitory potency has been observed for reduction of
nitrazepam or dantrolene in our previous studies [9,10], and the almost same results have also
been obtained in this study (Figs. 5G and H). We investigated the effects of allopurinol, a
potent inhibitor of human XOR [20], because XOR has a similar substrate specificity and
molecular structure to those of AOX1 [21] with a relatively high (approximately 50%) amino
acid homology and requirement for a molybdenum cofactor [22]. The reductase activities of
the 6 nitroaromatic drugs in addition to dantrolene and nitrazepam were not inhibited by
allopurinol. Thus, the involvement of XOR was excluded. These results supported that AOX1
is the enzyme responsible for the reduction of these nitroaromatic drugs in HLC.
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3.5. Structural characteristics of nitroaromatic drugs that can or cannot be reduced by human
AOX1
The structures of the nitroaromatic drugs used in this study are shown in Fig. 6 and are
categorized into two groups: those that were substantially reduced by human AOX1 or those
that were barely by human AOX1. As shown in Fig. 6A, the nitroaromatic drugs that were
reduced by human AOX1 tend to possess a relatively electron-deficient nitro group. For
example, flutamide and nilutamide have a strongly electron-withdrawing trifluoromethyl
group at the ortho position of the nitro group. Recently, it has been reported that
5-nitroquinoline, in which a nitro group is connected to an electron-deficient quinolone ring,
is reduced by AOX1 [19]. This result is consistent with the above theory. However, as shown
in Fig. 6B, the compounds that were not reduced by human AOX1 tend to have a relatively
electron-rich nitro group. For example, the nitro group in metronidazole is likely to be
relatively electron-rich due to electron donation from the N-1 atom in the imidazole ring. The
nitro group in tolcapone would also be relatively electron-rich due to electron donation from
the hydroxyl group at the ortho position of the nitro group. To definitively claim substrate
selectivity of AOX1 for nitro reduction, further studies with an increased number of
compounds are required. AOX1 can catalyze oxidation and hydrolysis [23]. AOX1 tends to
oxidize electron-rich carbon atoms on heteroaromatic rings or hydrolyze amide bonds if an
electron donating group is located at the ortho position of aniline [24]. The electron-rich
carbon atom in AOX1 substrates loses electrons when it is oxidized, and the electron-deficient
nitro group in AOX1 substrates receives electrons when it is reduced. These mechanisms are
chemically reasonable. This study provides new knowledge about the reduction catalyzed by
AOX1 in addition to oxidation and hydrolysis.
3.6. Significance of AOX1-mediated reduction of nitroaromatic drugs on pharmacokinetics
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and toxicity
Interestingly, all of the nitroaromatic drugs that were found to be reduced by AOX1 have
been reported to cause liver injury [25–32]. Because hydroxylamines formed via the reduction
of nitroaromatic drugs may cause hepatotoxicity, AOX1-mediated reduction of nitroaromatic
drugs is a concern because of their toxicity. Arylamine metabolites of the nitroaromatic drugs
that were reduced by human AOX1 that were identified in this study were detected in blood
[33–39]. Thus, the arylamine metabolites detected in humans would be produced by AOX1.
By contrast, there are no reports of detectable reduced forms in human urine or blood for
azelnidipine, metronidazole, nifedipine, and nimodipine, which were not reduced by AOX1.
This study demonstrated that tolcapone, which is well-known to cause liver injury, was not
reduced in HLM or HLC or by recombinant AOX1 (Figs. 2 and 3M), but its reduced form,
aminotolcapone, was detected in urine [39]. Therefore, tolcapone can be reduced by intestinal
bacteria, enzyme(s) in organs other than liver, or enzymes that require cofactors other than
MNA and NADPH in the liver.
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4.
Conclusion
We examined the substrate selectivity of human AOX1 in the reduction of nitroaromatic
drugs and found that compounds with a relatively electron-deficient nitro group were reduced
by human AOX1. This study provides fundamental information about the substrate selectivity
of human AOX1, and this information will be helpful to predict the possible toxicities of
nitroaromatic drugs.
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Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the
Japan Society for the Promotion of Science (16K08367), Kanazawa University SAKIGAKE
project 2018, and The National Institute for General Medical Sciences (GM100874) to jpj.
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Figure legends
Fig. 1. Possible metabolic pathways of nitroaromatic compounds in human.
Fig. 2. Reductase activities of 13 nitroaromatic drugs by recombinant human AOX1 in the
presence of 2 mM MNA. Recombinant human AOX1 (67.5 nM) was incubated with
nitroaromatic drugs (100 µM) for 3 hrs. Each column represents the mean ± SD of triplicate
determinations. ND: Not detected.
Fig. 3. Reductase activities of 13 nitroaromatic drugs by HLM or HLC in the presence or
absence of an NADPH-generating system or 2 mM MNA. HLM or HLC (0.5 mg/mL) were
incubated with nitroaromatic drugs (100 µM) for 3 hrs. Each column represents the mean ±
SD of triplicate determinations. ND: Not detected. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 4. Kinetic analyses of the reductase activities of 6 nitroaromatic drugs by HLC. Open and
closed circles represent the activities in the absence and presence of MNA, respectively. Each
point represents the mean ± SD of triplicate determinations.
Fig. 5. Inhibitory effects of several inhibitors on the reductase activities in HLC. The
inhibitors used in this study were as follows: estradiol, estrone, and raloxifene: known as
strong inhibitors of AOX1 [17,18]. Ketoconazole, promazine, and simvastatin: moderate
inhibitors. Diclofenac, metronidazole, and naloxone: hardly inhibit AOX1. All compounds
were used at the concentration of 50 µM. The control activities were 10.4 ± 0.4 pmol/min/mg
protein (clonazepam: 25 µM), 2.96 ± 0.5 pmol/min/mg protein (flunitrazepam: 60 µM), 56.6
± 7.1 pmol/min/mg protein (flutamide: 50 µM), 8.3 ± 0.6 pmol/min/mg protein (nilutamide:
200 µM), 33.3 ± 4.1 pmol/min/mg protein (nimesulide: 250 µM), 31.4 ± 1.6 pmol/min/mg
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protein (nimetazepam: 40 µM), 24.4 ± 0.2 pmol/min/mg protein (dantrolene: 60 µM), and
11.7 ± 0.5 pmol/min/mg protein (nitrazepam: 25 µM). Each column represents the mean ± SD
of triplicate determinations.
Fig. 6. Chemical structures of nitroaromatic drugs that (A) are or (B) are not reduced by
human AOX1.
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Table 1.
LS-MS/MS conditions for arylamines, reduced metabolites of nitroaromatic drugs.
Arylamine
Mobile phase
MRM
Collision energy
V
B concentration (%)
m/z
Aminoazelnidipine
Aminoclonazepam
Aminodantrolene
Aminoflunitrazepam
Aminoflutamide
40-80 (0–4 min), 80 (4-5 min), 40 (5-7 min)
30-70 (0–2 min), 70 (2–6 min), 30 (6–8 min)
10-90 (0-3 min), 90 (3-7.5 min), 10 (7.5-10 min)
50 (0–1 min), 95 (1-3 min), 50 (3-5 min)
40-90 (0–2 min), 90 (2–6 min), 40 (6–8 min)
20-70 (0–3 min), 70 (3-4 min), 20 (4-6 min)
30-70 (0–2 min), 70 (2–4 min), 30 (4-6 min)
30-70 (0–2 min), 70 (2–5.5 min), 30 (5.5–7.5 min)
30-70 (0–2 min), 70 (2–6 min), 30 (6–8 min)
30-70 (0–2 min), 70 (2–6 min), 30 (6–8 min)
40-80 (0–2 min), 80 (2–4 min), 40 (4-6 min)
30-70 (0–2 min), 70 (2–5 min), 30 (5–7 min)
30-70 (0–2 min), 70 (2–5 min), 30 (5-7 min)
553.30 > 167.00
286.10 > 121.10
285.15 > 186.00
284.00 > 135.05
247.10 > 227.10
141.90 > 98.05
317.10 > 224.05
288.10 > 268.05
279.05 > 200.00
266.15 > 135.05
389.20 > 313.05
252.30 > 121.20
244.10 > 119.05
34.0
32.0
18.0
28.0
17.0
Aminometronidazole
Aminonifedipine
Aminonilutamide
Aminonimesulide
Aminonimetazepam
Aminonimodipine
Aminonitrazepam
Aminotolcapone
19.0
12.0
18.0
20.0
28.0
9.0
28.0
16.0
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Table 2.
Kinetic parameters of each substrate of AOX1 in HLC in the absence or presence of MNA.
Substrate
MNA
Km
Vmax
pmol/min/mg protein
0.6 ± 0.1
42.5 ± 1.5
0.5 ± 0.1
30.1 ± 0.8
4.0 ± 0.2
148.9 ± 10.9
1.2 ± 0.1
41.2 ± 0.1
1.8 ± 0.1
65.0 ± 0.7
11.9 ± 2.0
55.2 ± 2.1
8.1 ± 0.2
207.4 ± 4.3
10.1 ± 0.2
40.2 ± 1.7
CLint
µL/min/mg protein
0.1 ± 0.0
1.5 ± 0.1
0.0 ± 0.0
0.5 ± 0.0
0.1 ± 0.0
2.4 ± 0.1
0.0 ± 0.0
0.2 ± 0.0
0.0 ± 0.0
0.2 ± 0.0
0.6 ± 0.1
1.3 ± 0.2
0.2 ± 0.0
3.8 ± 0.2
0.1 ± 0.0
2.3 ± 0.2
µM
-
+
-
6.7 ± 0.5
27.9 ± 1.6
46.0 ± 19.1
62.3 ± 2.3
53.0 ± 0.9
60.8 ± 6.7
38.3 ± 1.4
199.0 ± 10.9
201.4 ± 22.1
284.9 ± 28.7
18.5 ± 2.2
41.2 ± 7.7
45.4 ± 3.5
55.1 ± 3.2
122.2 ± 16.5
17.2 ± 0.4
Clonazepam
Flunitrazepam
Flutamide
+
-
+
-
Nilutamide
Nimesulide
Nimetazepam
Dantrolene
Nitrazepam
+
-
+
-
+
-
+
-
+
Data are mean ± SD.
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